Hybrid nanopore sensors

ABSTRACT

The disclosure provides detection apparatus having one or more nanopores, methods for making apparatus having one or more nanopore and methods for using apparatus having one or more nanopores. Uses include, but are not limited to detection and sequencing of nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 17/188,193filed Mar. 1, 2021 which is a division of U.S. application Ser. No.16/707,554 filed Dec. 9, 2019 now U.S. patent Ser. No. 10/961,576 issuedMar. 30, 2021 which is a continuation of U.S. application Ser. No.15/522,987 filed Apr. 28, 2017 now U.S. Pat. No. 10,519,499 issued Dec.31, 2019 which is the U.S. national phase of PCT/US2015/042680 filedJul. 29, 2015 and published in English as WO 2016019030 on Feb. 4, 2016which claims the benefit of U.S. Provisional Application No. 62,157,749,entitled “TETHERED NANOPORES, NANOPORES IN NANODISCS AND HYBRID NANOPORESENSORS” and filed May 6, 2015, and U.S. Provisional Application No.62/031,762, entitled “NANOPORES IN NANODISCS AND HYBRID NANOPORESENSORS,” filed Jul. 31, 2014, the disclosures of which are incorporatedherein by reference in their entireties for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with Government support under National Instituteof Health (NIH) Grant Number 5R21HG007833-02 awarded by the PublicHealth Services (PHS). The Government has certain rights in theinvention.

BACKGROUND

Protein nanopore devices have been explored for many sensorapplications, especially in the field of DNA/RNA sequencing.Discrimination of nucleotide bases has been demonstrated by reading theionic current signal when the DNA molecule is passing through thenanopore. Following from these initial observations, protein basednanopore devices have been postulated to have great potential to pushforward nucleic acid sequencing technology in terms of lower price, fastturn around and longer read length.

A more mature technology, sequencing-by synthesis (SBS), has made rapidprogress in the last five years, doubling throughput about every sixmonths, far outpacing Moore's law for the semiconductor industry. Thisrapid progress has been enabled by primarily three key improvements toSBS protocols: (1) cycle sequencing chemistry improvements; (2)increases in density of nucleic acid colonies on surfaces; and (3)improvement in imaging/scanning technology. These three technologicalimprovements have increased throughput of commercial systems from about1 Gb per run in January 2007 to over 1 Tb per run in June 2012. However,these technological advances have not been found to be directly portableto nanopore technologies.

In spite of this rapid progress in SBS improvement, 30× genome pricesare still over $1000/genome with turn-around times in excess of a week.Moreover, de novo assembly and haplotyping of human genomes obtained viaSBS is challenged by short reads. Strand sequencing via nanopores, canpotentially read up to 50,000 bases within a few minutes. This singlemolecule platform to date appears to achieve speed but at the cost ofgreatly decreased parallelization.

Parallelization of biological nanopores is notoriously difficult. Thefragility of the platform itself, especially the semi-fluidic nature ofthe lipid bilayer, demands specialized handling often by highly trainedtechnicians, making nanopore systems less practical for wide spreadcommercialization. The current technique for assembly of proteinnanopore devices is mostly manual, laborious and time consuming. Afterpainting a lipid bilayer over a substrate having an array of micrometersized apertures, the operator contacts the coated array with a solutionhaving a carefully titrated quantity of the protein. The amount ofnanopore is selected according to Poisson statistics to maximize thenumber of apertures that acquire a nanopore while minimizing the numberof apertures that are loaded with greater than one nanopore. After aspecific incubation period has lapsed, the nanopore-containing solutionis washed away. Poisson loading requires the time period to be carefullyselected to achieve the highest possible number of apertures having oneand only one nanopore. The results are highly dependent upon the skillof the operator and not easily adaptable to large scale devicemanufacturing.

Thus, a major challenge for nanopore technology is to increase therobustness of the platform and simultaneously improve parallelization,for example, in sequencing applications. The present disclosureaddresses this need and provides other advantages as well.

BRIEF SUMMARY

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

The present disclosure provides a hybrid nanopore consisting of aprotein nanopore inserted in a solid state nanopore. In particularembodiments, the hybrid nanopore is created with and ultimately includesprotein nanopores inserted in lipid nanodiscs. In accordance with themethods set forth herein the nanopore-containing lipid nanodiscs can beself-assembled.

In particular embodiments, a protein nanopore is initiallyself-assembled within a lipid nanodisc in free solution, as shown inFIG. 1A; then the protein nanopore is grafted with a nucleic acid asshown in FIG. 1B; then electrophoretic force is used to drive thenucleic acid chain through the solid-state nanopore until the proteinnanopore-containing nanodisc comes in contact with the solid statenanopore surface as shown in FIG. 1C.

Non-limiting advantages of the lipid nanodisc assisted nanopore assemblyscheme of the present disclosure, when compared to typical nanoporefabrication technologies that are based on suspended lipid membraneinclude, for example:

-   -   (1) Structural stability: the protein nanopore is inserted in a        hybrid membrane and the solid nanopore supports most of the        membrane and provides a nanometer-sized aperture.    -   (2) Scalable fabrication: the nature of the interactions between        the protein nanopore-containing lipid nanodisc and the solid        state nanopore are self-limiting such that fabrication is far        less fickle than assembly methods that rely on Poisson loading        of nanopores into solid-state apertures. Insertion of a single        protein nanopore into the bilayer can be accomplished during        reconstitution into a lipid nanodisc. The lipid nanodisc's size        (e.g. 10-13 nm in some embodiments) can be selected to favor        incorporation of only a single protein nanopore (e.g. ˜5 nm Msp        pore). Furthermore, the solid state nanopore size (e.g. 7-10 nm        diameter aperture in some embodiments) can be selected to allow        for assembly of only one protein-nanodisc complex per        solid-state nanopore aperture. Thus in many embodiments, there        is no need for active monitoring of electrical characteristics        during assembly and screening of multiple insertions.    -   (3) Better electrical insulation: direct insertion of a protein        nanopore in a solid-state nanopore typically requires detailed        matching of the size and shape between the two nanopores to        achieve adequate ionic current isolation. In the presently        disclosed methods, the lipid nanodisc can merely be selected to        be larger than the entrance of the solid state nanopore. Current        leakage can be prevented by a wide range of nanodisc sizes above        a minimum value such that avoiding current leakage will no        longer require detailed shape matching between protein and solid        state nanopores.    -   (4) Relaxed fabrication requirements for solid state nanopores:        solid-state nanopores having pore diameter greater than 5 nm can        form functional hybrid nanopores with protein        nanopore-containing lipid nanodiscs. This feature size is        readily achievable by state-of-art semiconductor technology,        whereas leading-edge pore fabrication, required for creating        solid state nanopores having diameters in the 2-3 nm range        required for direct insertion of protein nanopores, is highly        variable, costly and overly dependent on operator skill.    -   (5) Allows massive parallelization: elimination of micron-sized        lipid membranes can allow a solid-state nanopore platform to        achieve a high degree of parallelization via integration with        on-chip amplifiers and data acquisition circuits. Exemplary        amplifiers, circuits and other hardware useful for data        acquisition are described in U.S. Pat. Nos. 8,673,550 or        8,999,716; Rosenstein et al., Nano Lett 13, 2682-2686 (2013); or        Uddin et al., Nanotechnology 24, 155501 (2013), each of which is        incorporated herein by reference.    -   (6) Lower intrinsic noise: solid-state nanopores typically        require proper wetting for stable transconductance. Since the        solid-state nanopore is only utilized as a mechanical support in        particular embodiments of the system set forth herein those        embodiments will be expected to exhibit lower noise than a        standalone solid-state nanopore.

The present disclosure describes methods for making hybrid nanoporescombining top-down patterning and nanofabrication of solid-statenanopores with bottom-up biological assembly of protein nanopores.Existing techniques for nucleic acid sequencing with nanopore,especially biological nanopores in lipid bilayers, achieve high speedand read length at the cost of greatly decreased parallelization anddata throughput. The fragility of the existing platforms also make themless practical in commercialization. The compositions and methods of thepresent disclosure can overcome these limitations by establishing ahybrid biological-solid state structure that can be self-assembled in arobust automated manner with high efficiency.

As set forth in further detail herein, lipid-based carriers ofbiological channels can be assembled at solid-state nanopores havingdiameters greater than 10 nm. Methods set forth herein can be used forinsertion of nanopore proteins in lipid nanodiscs, self-limited assemblyof the protein nanopore-nanodiscs with solid-state nanopores, andanalytical applications of the hybrid platform, such as nucleic aciddetection and sequencing. The resulting platform can be configured toprovide the speed and read length characteristics ofprotein-nanopore-based nucleic acid sequencing systems while providinggreater parallelization and system stability available from the use ofestablished semiconductor technologies.

The present disclosure also provides tethered nanopores. In particular,nanopores can be tethered to electrodes or other solid supports inaccordance with the present disclosure.

In a particular embodiments, the present disclosure provides a detectionapparatus that includes (a) an electrode; (b) a nanopore tethered to theelectrode; and (c) a membrane surrounding the nanopore. Multiplexembodiments are also provided. For example a detection apparatus of thepresent disclosure can include (a) a plurality of electrodes; (b) aplurality of nanopores, each of the nanopores tethered to an electrodein the plurality of electrodes; and (c) a membrane surrounding each ofthe nanopores.

Also provided is a method of making a detection apparatus. The methodcan include the steps of: (a) providing a solid support having an arrayof electrodes; (b) providing a plurality of nanopores; (c) contactingthe plurality of nanopores with the array of electrodes to attachindividual nanopores to individual electrodes via a first tether,thereby making an array of nanopores that are tethered to electrodes inthe plurality of electrodes; and (d) contacting the array of nanoporeswith membrane material to form a membrane that surrounds each of thenanopores in the array.

Non-limiting advantages of particular embodiments set forth herein forassembly of detection apparatus using tethered nanopores include, forexample:

-   -   (1) Increased levels of spatial control over nanopore deposition        due to the ability to exploit steric exclusion such that one and        only one nanopore is accommodated at each electrode.    -   (2) Highly parallel assembly of multiple nanopores into a        detection apparatus.    -   (3) Ability to achieve nanopore loading efficiencies that beat        the limitations of Poisson statistics that hinder standard        methods of loading nanopores into multiplex detectors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a diagrammatic representation of a protein nanopore thatis initially self-assembled within a lipid nanodisc in free solution;FIG. 1B shows the protein nanopore grafted with a nucleic acid; FIG. 1Cshows assembly of the protein nanopore-containing nanodisc with thesolid-state nanopore due to the nucleic acid chain being driven throughthe solid-state nanopore until the nanodisc lipids come in contact withthe solid state surface.

FIG. 2A shows a diagrammatic representation of a 7-10 nm solid-statenanopore assembled with a lipid nanodisc that in turn has a proteinnanopore inserted in the lipid membrane, wherein a nucleic acid isgrafted to the protein nanopore; FIG. 2B shows a diagrammaticrepresentation of a 10-13 nm lipid nanodisc (light grey) bounded by ascaffold protein (dark ring).

FIG. 3 shows a diagrammatic representation of a process for lipidnanodisc formation.

FIG. 4 shows a diagrammatic representation of a lipid nanodisc loadingtechnique in which His-tagged protein (e.g. a porin can be used in placeof CYP3A4) is added to the initial mixture, and a metal chelating columnis used to separate protein-loaded discs from blank discs.

FIG. 5 shows fractions from expression, reconstitution and purificationof His-tagged MspA loaded on a polyacrylamide gel. Lane M, proteinladders; Lane S, soluble fraction of cell lysate; Lane F, flow throughfrom Ni-NTA spin column; Lane W, first Wash fraction; Lane E, elution ofHis-tagged MspA.

FIG. 6A shows a solid-state nanopore test fixture layout and FIG. 6Bshows an implementation of the layout. A nanopore chip (“Si chip”) isinserted at the indicated location, and sealed in place by O-ring rubbergasket. Ag/AgCl electrodes on the cis and trans side of the pore providethe current source. The current is measured with an Axopatch 200Bamplifier.

FIG. 7 shows biopore translocation measurement of a 91-mer DNAtranslocated through MspA biopore inserted into a suspended membrane.The vertical axis is normalized to pore open-state current.

FIG. 8 shows a reaction in which a lipid nanodisc is incubated withcholesterol-TEG DNA to produce a DNA tether on the nanodisc. The DNAtether can be used to electrophoretically pull the lipid nanodisc over asolid state nanopore.

FIG. 9 shows a method for enhanced coupling between a lipid nanodisc anda solid state nanopore. The Si₃N₄ surface of the solid support is firsttreated with an organosilane, followed by a cholesterol derivative.

FIG. 10 shows results of optimization of a protocol for inserting ofMspA into a lipid nanodisc.

FIG. 11A shows a TEM image of the high molecular weight elution fractionfrom the middle trace of FIG. 10 .

FIG. 11B shows a TEM image of the low molecular weight elution fractionfrom the middle trace of FIG. 10 . Sites of MspA insertion are indicatedwith arrows.

FIG. 12 shows a diagrammatic representation of a method for tethering ananopore to an electrode, wherein membrane tethers are attached to theelectrode after the nanopore is attached to the electrode.

FIG. 13 shows a diagrammatic representation of a method for tethering ananopore to an electrode, wherein membrane tethers are attached to theelectrode before the nanopore is attached to the electrode.

FIG. 14 shows a diagrammatic representation of a method for tethering ananopore to an electrode, wherein nanopore tethers are attached to theelectrode before the electrode is contacted with the nanopore.

DETAILED DESCRIPTION

The present disclosure provides a hybrid nanopore system based upon asolid-state nanopore sealed by a protein-pore embedded lipid nanodisc. Aconceptual illustration of a particular embodiment is illustrated inFIG. 2 .

Current nanopore strand sequencing platforms are based upon freediffusion and insertion of a protein nanopore (also referred to hereinas a “biological pore” or “biopore”) into a suspended micron sizedlipid-bilayer. Various methods have been developed to detect singleinsertion events, either by monitoring the ionic current or the ACimpedance. These approaches rely on additional monitoringcircuits/programs which can enlarge the device footprint and increasesystem complexity. The micron-size lipid bilayer is susceptible tomechanical vibration, adding complexity to device fabrication, shipment,storage and operation. Alternatively, solid-state nanopores with sizesgreater than 10 nm are robust and compatible both with high through-putmanufacturing and operation, providing a useful platform for nanoporetechnology. As such, embodiments of the present disclosure utilize solidstate nanopores as a platform to assemble single protein nanopores withlipid nanodisc as a rim seal to the solid state nanopore.

Particular embodiments employ insertion of a protein nanopore into alipid nanodisc carrier and apply electrophoretic force, possibly with aDNA tether, to guide the protein/lipid complex to the solid statenanopore. A lipid nanodisc can be, for example, a 7 to 13 nm-diameterlipid bilayer disc that is optionally stabilized by membrane scaffoldprotein (MSP), a derivative of apoA-I. It will be understood that ananodisc can have a diameter that is smaller than 7 nm (e.g. smallerthan 6 nm, 5 nm, 4 nm, 2 nm or less in diameter) or larger than 13 nm(e.g. larger than 15 nm, 20 nm, or 25 nm or greater in diameter).Typically, the area of lipid disc used in a method or composition setforth herein is no greater than about 50,000 nm² or in some cases nogreater than about 10,000 nm² or sometimes no greater than about 1,000nm² or even other times no greater than about 500 nm².

As exemplified in FIG. 2B, the lipid nanodisc can be composed of abilayer of lipid molecules surrounded by two parallel belt-like MSPs inwhich the amphipathic helices of the MSPs stabilize the hydrophobicfatty acid on the edge of the lipid disc. Particularly useful lipidnanodiscs and compositions and methods for their manufacture are setforth, for example, in Nath et al., Biochemistry 46, 2059-2069 (2007,),which is incorporated herein by reference.

Lipid nanodiscs can be prepared by mixing MSP with detergent stabilizedphospholipid. Self-assembly of nanodiscs occurs during removal of thedetergent from the mixture, as set forth below. It has been demonstratedthat the presence of MSP confines the shape and size of the lipidnanodisc and provides a narrow size distribution (±3%), excellentreproducibility and exceptional stability in detergent-free aqueoussolution. The ratio of MSP to detergent can be selected to achievedesired size and characteristics of the nanodiscs. For example, thenumber of structural units of the MSP can be varied to allow thenanodisc diameter to be tuned from 9.8 nm to 12.9 nm as set forth inDenisov et al., J. Am. Chem. Soc. 126, 3477-3487 (2004), which isincorporated herein by reference. These diameters are well-matched tosolid-state nanopores that can be reliably fabricated with commerciallyavailable methods. Nanodiscs can be an effective carrier to incorporatemembrane proteins. A number of membrane proteins have been integratedinto nanodiscs such as cytochrome, seven-transmembrane segment proteins,bacterial chemoreceptors and human mitochondrial voltage-dependent anionchannel protein. Exemplary methods for incorporating membrane proteinsinto nanodiscs are set forth in Raschle et al., J. Am. Chem. Soc. 131,17777-17779 (2009). Similar methods can be used to insert proteinnanopores, such as α-hemolysin, MspA, Cytolysin and others into lipidnanodiscs. Further examples, of useful protein nanopores are set forthin U.S. Pat. No. 8,673,550, which is incorporated herein by reference.

Coupling between the lipid nanodisc and the solid state nanopore can befacilitated by chemically engineering the lipid/solid interface. Forexample, the solid surface can be functionalized with cyanopropylsilane, cholesterol, RAFT polymerization, or other materials that bindto or adhere to lipids and/or MSP. Exemplary materials and methods forfunctionalization of the surfaces are set forth in White et al., J AmChem Soc 129, 11766-11775 (2007) and Kwok et al., PLOS One DOI:10.1371/journal.pone.0092880 (2014), each of which is incorporatedherein by reference. Similarly, the lipid nanodisc (MSP or lipid) can beengineered to contain coupling or adapter molecules that enable matingwith the functionalization on the surface of the solid state nanopore.Such engineering can provide greater than 100 GΩ insulation between thelipid nanodisc and the solid state nanopore. These levels of insulationcan facilitate high levels of base discrimination for nucleic acidtranslocation and sequencing applications. Lipid nanodiscs can also becoupled to a solid state nanopore using reagents and methods set forthherein in the context of tethering lipids to electrodes. Accordingly,tethers that are used to attach lipids to electrodes can be used tocouple a nanodisc to a solid state pore.

In particular embodiments, the process for lipid nanodisc formation canbe carried out as exemplified in FIG. 3 and further detailed in Baas“Characterization of monomeric human cytochrome P450 3A4 and CytochromeP450 Reductase in nanoscale phospholipid bilayer discs”, University ofIllinois at Urbana-Champaign, U.S.A. (2006), ISBN 0542987236,9780542987236A, which is incorporated herein by reference. As shown inthe figure, Membrane Scaffold Protein can be self-assembled withdetergent-solubilized phospholipids to form nanodiscs. The self-assemblyoccurs as the detergent is removed, for example, using Bio-Beads®(Bio-Rad, Hercules CA). The product of the assembly reaction can then bepurified with size-exclusion chromatography. Optimization of thelipid/protein ratio results in mono-dispersed nanodiscs with sizedetermined by the length of the membrane scaffold protein.

Insertion of single and even multiple proteins in the nanodiscs can beachieved via an additional purification step that uses a nickel-basedaffinity matrix to specifically bind a polyhistidine affinity tag on thenanopore protein, for example, using methods similar to thosedemonstrated in Baas “Characterization of monomeric human cytochromeP450 3A4 and Cytochrome P450 Reductase in nanoscale phospholipid bilayerdiscs”, University of Illinois at Urbana-Champaign, U.S.A. (2006), ISBN0542987236, 9780542987236A, which is incorporated herein by reference. Adiagrammatic representation of a useful method for affinity-basedseparation of biopore-bearing nanodiscs is shown in FIG. 4 .

Biological pore mutagenesis can be used to optimize protein pores forassembly or use in a composition or method set forth herein. As setforth above, the process of incorporating a protein channel in thenanodisc can benefit from a polyhistidine affinity tag on the proteinand from purification of the mixed nanodisc population over a Niaffinity column. Protein engineering and mutagenesis techniques can beused to mutate biological pores and tailor their properties for specificapplications. MspA with a C-terminal 6×His tag can be expressed,reconstituted and purified as demonstrated by the SDS-PAGE gel shown inFIG. 5 (Lane M, protein ladders; Lane S, soluble fraction of lysate fromcells expressing His-tagged MspA; Lane F, flow through from Ni-NTA spincolumn; Lane W, 1st Wash fraction from Ni-NTA spin column; Lane E,elution of His-tagged MspA from Ni-NTA spin column). Biologicalfunctionality of the purified His-tagged MspA was found to be similar tothat of a non-tagged MspA. Other mutations can also be introduced forpurification purposes. For example, a cysteine moiety can be introducedinto the protein sequence by mutagenesis and used for chemicalconjugation to thiol reactive moieties (e.g. maleimides oriodoacetamides) of affinity tags. Exemplary affinity tags include biotin(which can mediate purification via solid-phase streptavidin), DNA andRNA (which can mediate purification via solid-phase nucleic acids havingcomplementary sequences), epitopes (which can mediate purification viasolid-phase antibodies or antibody fragments) or other ligands (whichcan mediate purification via solid-phase receptors for those ligands).

Affinity tags (e.g. His-tags and others set forth herein) and chemicalconjugation techniques (e.g. modifications of cysteines set forth above)can be used to attach tethers to nanopores for use in a variety ofmethods, compositions or apparatus set forth herein. For example, theresulting tethers can be used to attract or attach a nanopore containingnanodisc at or near a solid state nanopore. The tethers can also be usedto attach a nanopore to an electrode or other solid-phase support.

The present disclosure provides a system for data acquisition using oneor more hybrid protein-solid state nanopores. An exemplary system isshown in FIG. 6 . In this example, the solid-state nanopore is insertedat the indicated location, and sealed in place by an o-ring. StandardAg/AgCl electrodes on the cis and trans-side of the pore provide thecurrent source. Data from the nanopore can be acquired with an Axopatch200B amplifier. Preliminary data from a solid self-supporting SiNmembrane (no nanopore) has demonstrated greater than 100 GΩ resistance,indicative of a robustly sealed flowcell (less than 0.9 pA leakagecurrent at 200 mV bias). This type of setup is consistent withwell-established “standard” setups used to evaluate analyticalcapabilities in the nanopore art. Representative data from a 91nucleotide DNA that was translocated through a biological nanopore instrand-displacement mode is shown in FIG. 7 .

The disclosure provides a detection apparatus having (a) a solid supportincluding an array of solid state nanopores; (b) a plurality of lipidnanodiscs on the surface of the solid support, wherein each of the lipidnanodiscs forms a seal at each of the solid state nanopores, and whereinthe lipid nanodiscs are separated from each other by interstitialregions on the surface of the solid support; and (c) a plurality ofprotein nanopores inserted in the lipid nanodiscs to create apertures ineach of the seals.

The detection apparatus can further include electrodes embedded in thesolid support. The electrodes can be used to monitor assembly ofnanodiscs and/or protein nanopores into the solid state nanopores. Theelectrodes can also be used for data collection during analyte detectionsteps. Electrodes used for monitoring and detection need not be embeddedin the solid support and can instead be provided in a separateapplication-specific integrated circuit (ASIC) chip, for example.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid and incapable of passing a liquidabsent an aperture. Exemplary solid supports include, but are notlimited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles, andpolymers. Particularly useful solid supports comprise modified siliconsuch as SiN membranes on Si substrate. For some embodiments, solidsupports are located within a flow cell apparatus or other vessel.

As used herein, the term “solid state nanopore” refers to a hole thatpasses through a solid support to allow passage of a liquid or gas. Asolid state nanopore will generally have a lumen or aperture that isbetween about 1 nm and 1 μm in diameter. However, larger or smallerdiameters are possible. A solid state nanopore is generally made frommaterials of non-biological origin. A solid state pore can be ofinorganic or organic materials. Solid state pores include, for example,silicon nitride pores, silicon dioxide pores, and graphene pores.

As used herein the term “lipid nanodisc” refers to a lipid bilayer sheetthat occupies an area between about 3 nm² and 3 μm². For example, thearea occupied can be at least about 5 nm², 10 nm², 50 nm², 100 nm²,1,000 nm², 10,000 nm², 100,000 nm², or more. Alternatively oradditionally, the area occupied can be at most about 100,000 nm², 10,000nm², 1,000 nm², 100 nm², 50 nm², 10 nm², 5 nm², or less. A lipidnanodisc can, but need not necessarily, occupy a circular area. Thecircular area occupied by a nanodisc can have a diameter between about 1nm and 1 μm. However, larger or smaller areas or diameters are possible.A lipid nanodisc can, but need not necessarily, be flat or planar. Inparticular conditions a lipid nanodisc can be distinguished from avesicle or liposome due to the absence of an aqueous lumen for thenanodisc and can be distinguished from a micelle due to the presence ofa bilayer in the nanodisc. Several embodiments are exemplified hereinusing lipid nanodiscs. It will be understood that a nanodisc can be madefrom other materials as well. For example, a nanodisc can be formed froma non-lipid membrane such as those set forth below.

As used herein the term “membrane” refers to a sheet or other barrierthat prevents passage of electrical current or fluids. The membrane istypically flexible or compressible in contrast to solid supports setforth herein. The membrane can be made from lipid material, for example,to form a lipid bilayer, or the membrane can be made from non-lipidmaterial. The membrane can be in the form of a copolymer membrane, forexample, formed by diblock polymers or triblock polymers, or in the formof a monolayer, for example, formed by a bolalipid. See for example,Rakhmatullina et al., Langmuir: the ACS Journal of Surfaces and Colloids24:6254-6261 (2008), which is incorporated herein by reference.

As used herein, the term “interstitial region” refers to an area in asubstrate or on a surface that separates other areas of the substrate orsurface. For example, an interstitial region can separate one nanoporeof an array from another nanopore of the array. The two regions that areseparated from each other can be discrete, lacking contact with eachother. In many embodiments the interstitial region is continuous whereasthe features are discrete, for example, as is the case for an array ofpores or apertures in an otherwise continuous surface. The separationprovided by an interstitial region can be partial or full separation.Interstitial regions will typically have a surface material that differsfrom the surface material of the features on the surface. For example,features of an array can have an amount or concentration of lipidmaterial or protein material that exceeds the amount or concentrationpresent at the interstitial regions. In some embodiments the lipidmaterial or protein material may not be present at the interstitialregions.

The interstitial regions can separate lipid nanodiscs by at least 1 nm,5 nm, 10 nm, 100 nm, 1000 nm or more in an apparatus set forth herein.Alternatively or additionally, the separation may be no more than 1000nm, 100 nm, 50 nm, 10 nm or 5 nm.

As used herein the term “protein nanopore” refers to a polypeptide,formed as one or more subunits, to create an aperture through a membraneor solid support. Exemplary protein nanopores include α-hemolysin,Mycobacterium smegmatis porin A (MspA), gramicidin A, maltoporin, OmpF,OmpC, PhoE, Tsx, F-pilus, SP1 (Wang et al., Chem. Commun., 49:1741-1743,2013) and mitochondrial porin (VDAC)XX, Tom40, (U.S. Pat. No. 6,015,714and Derrington et al., Proc. Natl. Acad. Sci. USA, 107:16060 (2010)).“Mycobacterium smegmatis porin A (MspA)” is a membrane porin produced byMycobacteria, allowing hydrophilic molecules to enter the bacterium.MspA forms a tightly interconnected octamer and transmembranebeta-barrel that resembles a goblet and contains a central channel/pore.Other useful pores are set forth in U.S. Pat. No. 8,673,550. Each of theabove nanopore references is incorporated herein by reference.

As used herein, the term “hybrid nanopore” is intended to mean anaperture that is made from materials of both biological andnon-biological origins, extending across a barrier such as a membranefor example, that permits hydrated ions and/or water soluble moleculesto cross from one side of the barrier to the other side of the barrier.Materials of biological origin are defined above and include, forexample, polypeptide and polynucleotide. A biological and solid statehybrid pore includes, for example, a polypeptide-solid state hybrid poreand a polynucleotide-solid state pore.

As used herein the term “tethered” is intended to refer to the state oftwo items being attached through a linker moiety. The attachment can becovalent, for example, such that an unbroken chain of covalent bondslinks the two items. Alternatively, the attachment can be mediated by atleast one non-covalent bond, such as a specific binding interactionbetween a receptor and ligand. Exemplary receptor-ligand pairs include,without limitation, streptavidin (and analogs thereof) and biotin (oranalogs thereof); antibody (or functional fragments thereof) andepitopes; lectins and carbohydrates; complementary nucleic acids,nucleic acid binding proteins and their nucleic acid substrates.

A “tether” is a linker moiety that can optionally be used to attach twoitems. In some configurations a tether is attached to a single item,such as a nanopore, lipid, membrane material or solid support. A tetherthat is attached to one item can optionally be attached to a second itemor used for other purposes. A tether can include nucleotides or nucleicacid material. Alternatively, a tether can be made of a material otherthan nucleic acid material or can be devoid of nucleotides.

In particular embodiments, a detection apparatus can include solid-statenanopores that form apertures between reservoirs that are separated bythe solid support.

In particular embodiments, a seal that is formed over a solid statenanopore prevents flow of fluids and/or flow of electrical current.However, a protein nanopore can form an aperture in the seal.

A protein nanopore can occupy an area on the surface of a lipid nanodiscthat is at least about 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm or more indiameter. Alternatively or additionally, a protein nanopore can occupyan area on the surface of a lipid nanodisc that is at most about 1micron, 500 nm, 100 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5, nm or less.

In particular embodiments, a detection apparatus can include at least10, 100, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, or more solid state nanopores.

In particular embodiments, a detection apparatus can include lipidnanodiscs covering least 10, 100, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, or moresolid state nanopores. No matter the total number of solid statenanopores, lipid nanodiscs can cover at least 10%, 25%, 50%, 75%, 90%,95%, 99% or more of the solid state nanopores of a detection apparatus.Accordingly, a detection apparatus can include at least 10, 100, 1×10³,1×10⁴, 1×10⁵, 1×10⁶, or more nanodiscs.

In particular embodiments, a detection apparatus can include proteinnanopores in the lipid nanodiscs covering least 10, 100, 1×10³, 1×10⁴,1×10⁵, 1×10⁶, or more solid state nanopores. No matter the total numberof solid state nanopores, protein nanopores can be inserted into lipidnanodiscs that cover at least 10%, 25%, 50%, 75%, 90%, 95%, 99% or moreof the solid state nanopores of a detection apparatus. Accordingly, adetection apparatus can include at least 10, 100, 1×10³, 1×10⁴, 1×10⁵,1×10⁶, or more nanodiscs having inserted nanopores.

A detection apparatus of the present disclosure can be used to detectany of a variety of analytes including, but not limited to, ions,nucleic acids, nucleotides, polypeptides, biologically active smallmolecules, lipids, sugars or the like. Accordingly, one or more of theseanalytes can be present in or passed through the aperture of a proteinnanopore in an apparatus set forth herein.

In particular embodiments, a detection apparatus can include a cisreservoir in contact with the array of solid-state nanopores and a transreservoir in contact with the array of solid state nanopores. The cisand trans reservoirs can contain electrodes located to apply a currentthrough the apertures formed by the protein nanopores. A cis reservoir,trans reservoir or both can be configured to maintain a liquid in bulkfluid communication with a plurality of nanopores in an apparatus setforth herein. Alternatively, one or both of the reservoirs may be incontact with only one or only a subset of the nanopores found in anarray or apparatus set forth herein.

In particular embodiments, each of the lipid nanodiscs that covers asolid state nanopore of an array set forth herein will have no more thanone protein nanopore inserted therein. However, it is also possible tomake and use an apparatus having more than one protein nanopore insertedper solid state nanopore. Similarly, individual nanodiscs, whethercovering a solid state nanopore or not, can include no more than oneprotein nanopore. Alternatively, individual nanodiscs can include morethan one protein nanopore.

A lipid nanodisc can be made from any of a variety of membranes orlipids. Suitable lipid bilayers and methods for making or obtaininglipid bilayers are well known in the art and disclosed in, for example,US 2010/0196203 and WO 2006/100484, each of which is incorporated hereinby reference. Suitable lipid bilayers include, for example, a membraneof a cell, a membrane of an organelle, a liposome, a planar lipidbilayer, and a supported lipid bilayer. A lipid bilayer can be formed,for example, from two opposing layers of phospholipids, which arearranged such that their hydrophobic tail groups face towards each otherto form a hydrophobic interior, whereas the hydrophilic head groups ofthe lipids face outwards towards the aqueous environment on each side ofthe bilayer. Lipid bilayers also can be formed, for example, by themethod of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69:3561-3566, incorporated herein by reference), in which a lipid monolayeris carried on aqueous solution/air interface past either side of anaperture which is perpendicular to that interface. The lipid is normallyadded to the surface of an aqueous electrolyte solution by firstdissolving it in an organic solvent and then allowing a drop of thesolvent to evaporate on the surface of the aqueous solution on eitherside of the aperture. Once the organic solvent has evaporated, thesolution/air interfaces on either side of the aperture are physicallymoved up and down past the aperture until a bilayer is formed. Othercommon methods of bilayer formation include tip-dipping, paintingbilayers, and patch-clamping of liposome bilayers. A variety of othermethods for obtaining or generating membranes are well known in the artand are equally applicable for use in the compositions and methods ofthe present disclosure (e.g. those pertaining to hybrid nanopores orthose pertaining to tethered nanopores). The reagents and methods setforth above can also be used in combination with tethered nanopores orin embodiments where membrane materials are tethered to an electrode orother solid support.

This disclosure further provides a method of making a detectionapparatus. The method can include steps of (a) providing a solid supporthaving an array of solid state nanopores; (b) providing a plurality oflipid nanodiscs, wherein the lipid nanodiscs include protein nanoporesinserted in the lipid; (c) contacting the plurality of lipid nanodiscswith the array of solid state nanopores to cover individual solid statenanopores of the array with one of the lipid nanodiscs. The method canbe used to make a detection apparatus having one or more of thecharacteristics set forth elsewhere herein.

In particular embodiments step (b) can further include incubating theprotein nanopores with lipids under conditions for inserting the proteinnanopores in the lipid nanodiscs. For example, the lipids can bedetergent solubilized and the conditions can include a technique forremoving the detergent from the protein nanopores.

As set forth elsewhere herein, the lipid nanodiscs can be electricallyattracted to the solid state nanopores (e.g. via electrophoresis).Optionally, the lipid nanodiscs can include charged tethers to mediatethe attraction. As one example, the charged tethers can be nucleicacids, in either naturally occurring form or as a non-naturallyoccurring analog form. The attachment site of the tether can be used toinsert nanodiscs with a particular orientation within the solid statenanopore. This allows the protein nanopore to be oriented in either aforward or reverse direction within the solid state nanopore. Mixes oftether attachment sites can generate any desired ratio of forward toreverse biological pores across an array of solid state nanopores.Similar control of nanopore orientation by placement of tethers can beachieved in embodiments where nanopore tethers are attached toelectrodes.

To enhance analyte capture from solution and facilitate two dimensionaldiffusion of analyte within the plane of the solid state nanoporemembrane into the vicinity of the biological nanopore, the solid statenanopore membrane can be derivatized with a compound that reversiblyinteracts with the analyte. The kinetic on/off rates between the analyteand membrane can be selected to allow a rapid random walk of the analyteacross the surface of the solid state nanopore membrane. Examples ofsuch interactions include a cholesterol tag interacting with a lipidmonolayer or bilayer, a DNA tag interacting with a DNA surface, andother interactions such as those set forth in Yusko et al., NatNanotechnol 6(4): 253-260 (2011). The interaction of the DNA tag withthe DNA surface can be facilitated by the use of recombinases that allowwalking by strand invasion across the surface of the solid statenanopore membrane.

In some embodiments of the methods, the quantity of the lipid nanodiscsthat is contacted with an array of solid state nanopores exceeds thequantity of solid state nanopores in the array. The lipid nanodiscs caninclude inserted protein nanopores. Thus, Poisson statistics need not berelied upon to obtain solid state nanopores with only one proteinnanopore. Instead, saturating amounts of the lipid nanodiscs (with orwithout inserted protein nanopores) can be contacted with an array ofsolid state nanopores. For example, the quantity of the lipid nanodiscs(with or without inserted protein nanopores) can exceed the quantity ofsolid state nanopores by at least 2 fold, 5 fold, 10 fold or more.

The present disclosure provides a detection apparatus that includes (a)an electrode; (b) a nanopore tethered to the electrode; and (c) amembrane surrounding the nanopore. Multiplex embodiments are alsoprovided. For example a detection apparatus of the present disclosurecan include (a) a plurality of electrodes; (b) a plurality of nanopores,each of the nanopores tethered to an electrode in the plurality ofelectrodes; and (c) a membrane surrounding each of the nanopores.

An electrode used in a method or apparatus of the present disclosure canbe made of any of a variety of materials used in nanopore detectiondevices or other devices for electrochemical processes. In particularembodiments, electrodes are made of a metal. Electrodes can be patternedon a solid support such that the support includes a plurality ofelectrodes. For example, the solid support can include at least 1, 2,10, 100, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, or more electrodes. Alternativelyor additionally, a solid support can include no more than 1×10⁶, 1×10⁵,1×10⁴, 1×10³, 100, 10, 2 or 1 electrodes.

Electrodes that are on a solid support can be separated from each otherby interstitial regions that lack the electrode material or areotherwise non-conductive. Accordingly, the electrodes can be spatiallyand functionally discrete. In some embodiments, each electrode islocated within a well or other concave feature on a solid support andthe walls of the concave features function as interstitial regionsbetween electrodes. Interstitial regions can separate electrodes on asolid support such that the average pitch (center-to-center) spacing ofthe electrodes is, for example, at least 5 nm, 10 nm, 25 nm, 50 nm, 100nm, 1 μm, 10 μm, 100 μm or more. Alternatively or additionally, thepitch can be, for example, no more than 100 μm, 10 μm, 1 μm, 100 nm, 50nm, 25 nm, 10 nm, 5 nm or less. In some embodiments, the interstitialregions can also lack lipids or nanopores or other materials describedherein as being attached to an electrode.

Electrodes used in an apparatus or method set forth herein can have anyof a variety of sizes or shapes that achieve a desired use of thedetection apparatus. For example, electrodes can have a footprint thatis round, rectangular, square, polygonal etc. The area occupied by anelectrode can be, for example, at least 25 nm², 100 nm², 500 nm², 1 μm²,50 μm², 100 μm², 1 mm 2, or larger. Alternatively or additionally, thearea of the electrodes can be, for example, at most 1 mm², 100 μm², 50μm², 1 μm², 500 nm², 100 nm², 25 nm², or smaller.

In particular embodiments, an electrode can be covered at leastpartially by a dielectric pad. Dielectric pads can provide reactivemoieties that interact with molecules, such as tether molecules, toachieve attachment of the molecules to the dielectric pad. Silanecoupling chemistry is particularly useful for coupling. Exemplarycoupling chemistries are set forth in US Pat App. Pub. Nos. 2014/0200158A1 or 2015/0005447 A1, each of which is incorporated herein byreference. Molecules that are attached to the dielectric pads will beeffectively attached to the electrode via the dielectric pad.

A dielectric pad used in a method or apparatus set forth herein can haveareas in the range exemplified above for electrodes. In some cases, thedielectric pad will occupy a fraction of the surface of an electrodethat is at most, 75%, 50%, 25%, 10%, 5%, 1% or less of the electrodesurface. Particularly useful dielectric pads will have an area that iscomparable to or smaller than the footprint of a nanopore. As such, thedielectric pad will have a capacity for one and only one nanopore. Theresulting steric exclusion allows an array of electrodes having thedielectric pads to be contacted with an excess of nanopores (i.e. anumber of nanopores that is larger than the number of electrodes in thearray) such that all or most of the electrodes can be attached tonanopores while avoiding the risk of attaching multiple nanopores atindividual electrodes that could occur if steric exclusion is notachieved.

Any of a variety of nanopores set forth herein or otherwise known in theart can be used in a tether attachment embodiment set forth herein.Particularly useful nanopores are protein nanopores such as Msp A.

A nanopore can be tethered to an electrode (e.g. via a dielectric pad)using covalent moieties or non-covalent binding moieties. An example ofa covalent attachment is when a nucleic acid tether is covalentlyattached to the nanopore and covalently attached to the dielectric pad.Other tethers can be similarly used for covalent attachment, includingfor example, non-nucleic acid tethers such as polyethylene glycol orother synthetic polymers. An example, of a non-covalent attachment iswhen a nanopore has an attached affinity moiety, such as a polyhistidine tag, Strep-tag or other amino acid encoded affinity moiety.Affinity moieties can bind non-covalently to ligands on a dielectric padsuch as nickel or other divalent cations that bind polyhistidine, orbiotin (or analogs thereof) that bind to Strep-tag. In some embodiments,such amino acid affinity moieties need not be used.

An apparatus set forth herein can include any number of tetherednanopores desired. For example, an apparatus can include at least 1, 2,10, 100, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, or more tethered nanopores.Alternatively or additionally, an apparatus can include no more than1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 100, 10, 2 or 1 tethered nanopore.

Any of a variety of membrane materials can be used in a nanoporetethering embodiment set forth herein. Exemplary lipids that can be usedinclude those set forth herein in the context of nanodisc embodiments orin references cited in context of those embodiments. Other useful lipidsmaterials are set forth in U.S. Pat. No. 8,999,716, which isincorporated herein by reference.

In particular embodiments, a membrane can be tethered to an electrode.The membrane can be tethered to an electrode to which a nanopore is alsotethered (e.g. via a dielectric pad). The tethers used to attachmembrane material to an electrode can be selected from those exemplifiedherein in regard to nanopore tethers. Tethers with reactive thiols canbe particularly useful for attaching to gold containing electrodes.Other tethers are also possible including, for example, tethers withsilane or phosphonate reactive groups which can be used to attach toconductive metal oxides (see for example Folkers et al., Langmuir 11:813(1995), which is incorporated herein by reference). In some embodiments,a different tether material is used for membrane material compared tothe tether used for a nanopore. Alternatively, the same tether type canbe used for both nanopores and membrane materials. It will be understoodthat in some embodiments, or in some stages of preparation of adetection apparatus, the membrane material that surround a nanopore neednot be tethered to the electrode to which the nanopore is tethered.

In some detection apparatus, particularly those with multipleelectrodes, the membrane that surrounds each of the nanopores can form aseal for the electrode to which the nanopore is tethered. The seal canbe made to prevent flow of fluids or electrical current from theelectrode to which the nanopore is tethered to other electrodes of thedetection apparatus. For example, the electrode can occur within a well(or other concave feature) and the membrane can seal the well by forminga continuous sheet that contacts the walls of the well. Thus a chamberis formed within the space defined by the electrode, the walls of thewell and the membrane. The chamber can function as a cis or transchamber depending upon the relative orientation of the electrodes andthe orientation of the inserted nanopore.

As set forth in further detail below, a detection apparatus havingtethered nanopores can be particularly useful for detection of nucleicacids including detection of nucleic acids in a sequencing method. Assuch, the detection apparatus can occur in a state where a nucleic acidanalyte is located in an aperture formed by the nanopore. The nucleicacid being an analyte that is to be detected is distinct and differentfrom a nucleic acid that may be used as a tether in the apparatus.

The detection apparatus can include other hardware used for detection ofnanopores, such as electrodes configured to create a current across amembrane and through a nanopore, an amplifier that is configured toamplify electrical signals generated at the nanopore, a computer coupledto the apparatus to evaluate signals detected from one or more nanoporesetc. Hardware useful for detecting signals from nanopores and that canbe modified for use in a method set forth herein is described in theart, for example, in U.S. Pat. Nos. 8,673,550 or 8,999,716; Rosensteinet al., Nano Lett 13, 2682-2686 (2013); or Uddin et al., Nanotechnology24, 155501 (2013), each of which is incorporated herein by reference.

Also provided is a method of making a detection apparatus. The methodcan include the steps of: (a) providing a solid support having an arrayof electrodes; (b) providing a plurality of nanopores; (c) contactingthe plurality of nanopores with the array of electrodes to attachindividual nanopores to individual electrodes via a first tether,thereby making an array of nanopores that are tethered to electrodes inthe plurality of electrodes; and (d) contacting the array of nanoporeswith membrane material to form a membrane that surrounds each of thenanopores in the array.

An exemplary embodiment of a method for tethering a nanopore to anelectrode is shown in FIG. 12 . A metal electrode is provided, thesurface of the electrode having a dielectric pad that covers a portionof the surface. For example, the dielectric pad can be an approximatelycircular pad that is about 10-50 nm in diameter. The dielectric pad canhave reactive silanes that are capable of forming a covalent bond with amoiety that is present on a nanopore tether. A nanopore can bechemically modified to include a nucleic acid tether having the moiety.The tether-containing nanopore can then be contacted with the dielectricpad under conditions for the silane to react with the tether tocovalently attach the nanopore to the dielectric pad. Once the nanoporehas been attached, membrane tethers can be reacted with the metalsurface of the electrode. For example, the membrane tethers can includethiols that form covalent bonds with the metal surface. In the last stepshown, membrane material (e.g. lipid material) can be contacted with thesystem under conditions to form a membrane (e.g. lipid bilayer) thatsurrounds the tethered nanopore and to form covalent bonds between themembrane tethers and lipids in the bilayer. Thus the membrane is alsotethered to the electrode.

Another embodiment is shown in FIG. 13 . The method uses similarcomponents to those used in FIG. 12 . However, here the membrane tethersare attached to the metal surface of the electrode prior to attachingthe nanopore.

A further exemplary embodiment is shown in FIG. 14 . In this embodiment,the metal electrode does not include a dielectric pad. The metalelectrode is reacted with membrane tethers in a first step. Then anano-printing technology, such as lithography, is used to attachnanopore tethers to the surface of the metal electrode. The nanoporetethers can be printed in a small area (e.g. approximately circularareas that are about 10-50 nm in diameter). A nanopore can then becontacted with the tether containing electrode under conditions tocreate a covalent linkage between the nanopore and tether. In the finalstep, a membrane (e.g. lipid membrane) can be formed around the tetherednanopore and reacted with the membrane tethers to become attached to theelectrode.

In particular embodiments, a plurality of nanopores is contacted with aplurality of electrodes in bulk. For example, a solution that includesthe nanopores can be contacted with a solid support having multipleelectrodes such that the nanopores in the solution are in fluidcommunication with multiple electrodes. The quantity of nanopores thatare contacted with the array of electrodes can exceed the quantity ofelectrodes in the array. This can be done, for example, to increase theprobability that most or all of the electrodes will become tethered to ananopore. The electrodes can be configured to have a capacity for nomore than one nanopore. For example, the electrode can have a surface orsurface portion that is relatively small compared to the size of thenanopore. As a result, once a first nanopore has attached to theelectrode a subsequent nanopore is sterically excluded from attaching tothe same electrode. Thus, an array can include a number of electrodeshaving a single nanopore that exceeds the number that would be expectedfrom typical Poisson statistics. For example, an array can be loadedwith a single nanopore at each of at least 50%, 65%, 75%, 90%, 95%, 99%or more of the electrodes in the array.

Nanopores that are contacted with membrane material can be detergentsolubilized. The detergent can then be removed from the proteinnanopores to allow a membrane (e.g. lipid bilayer) to surround thenanopores. For example, in situ dialysis can be carried out once thenanopores are attached to the electrode surface. Exemplary in situdialysis methods and tether compositions are set forth in Giess et al.,Biophysical J. 87:3213-3220 (2004), which is incorporate herein byreference.

In some embodiments, nanopores can be electrically attracted toelectrodes to facilitate attachment. A nanopore can be attracted basedon intrinsic charge. However, it is also possible to use a chargedtether, such as nucleic acid, and to attract the tether to theelectrode. Exemplary methods for electrically assisted localization ofnucleic acids to electrodes are set forth in U.S. Pat. No. 8,277,628,which is incorporated herein by reference.

Any of a variety of tethers described herein or known in the art can beused in a method set forth herein. A particularly useful tether is anucleic acid tether. In some embodiments, such as those exemplified inFIG. 12 and FIG. 13 , a tether can be attached to a nanopore prior toattaching the tether to an electrode. Alternatively, for example asshown in FIG. 14 , a nanopore tether can first be attached to anelectrode and then the nanopore is reacted with the nanopore tether tocreate an attachment between nanopore and tether.

A method set forth herein can include a step of attaching membranematerial to one or more electrodes via a tether. Particularly usefulmembrane tethers are bifunctional molecules having a lipophilic domainand a hydrophilic spacer. The lipophilic domain (e.g. phospholipid,cholesterol, or phytanyl) inserts into a membrane and the hydrophilicspacer can attach to a solid support, such as an electrode. Exemplarymembrane tethers are set forth, for example, in Giess et al.,Biophysical J. 87:3213-3220 (2004), which is incorporate herein byreference.

The present disclosure further provides a method of sequencing nucleicacids. The method can include steps of (a) providing a detectionapparatus having: (i) a solid support including an array of solid statenanopores; (ii) a plurality of lipid nanodiscs on the surface of thesolid support, wherein each of the lipid nanodiscs forms a seal at eachof the solid state nanopores, and wherein the lipid nanodiscs areseparated from each other by interstitial regions on the surface of thesolid support; and (iii) a plurality of protein nanopores inserted inthe lipid nanodiscs to create apertures in each of the seals; and (b)detecting passage, through the apertures in each of the seals, of (i) anucleic acid, (ii) a series of nucleotides removed from the nucleicacid, or (iii) a series of probes derived from nucleotides incorporatedinto the nucleic acid, thereby determining the sequence of the nucleicacid.

Also provided is a method of sequencing nucleic acids that includessteps of: (a) providing a detection apparatus that includes (i) aplurality of electrodes, (ii) a plurality of nanopores, each of thenanopores tethered to an electrode in the plurality of electrodes, and(iii) a membrane surrounding each of the nanopores; and (b) detectingpassage, through each of the nanopores, of (i) a nucleic acid, (ii) aseries of nucleotides removed from the nucleic acid, or (iii) a seriesof probes derived from nucleotides incorporated into the nucleic acid,thereby determining the sequence of the nucleic acid.

A nucleic acid detected in the methods of the present disclosure can besingle stranded, double stranded, or contain both single stranded anddouble stranded sequence. The nucleic acid molecules can originate in adouble stranded form (e.g., dsDNA) and can optionally be converted to asingle stranded form. The nucleic acid molecules can also originate in asingle stranded form (e.g., ssDNA, ssRNA), and the ssDNA can optionallybe converted into a double stranded form. Exemplary modes oftranslocating polynucleotides through a pore are set forth in WO 2013057495, which is incorporated herein by reference.

In some embodiments, sequencing can be carried out by passing a nucleicacid through a protein nanopore and detecting electrical signalsindicative of the passage of a particular nucleotide or series ofnucleotides (e.g. a “word” consisting of 2, 3 4, 5 or more nucleotides).In some embodiments, a certain level of controlled translocation of apolynucleotide through a nanopore can be achieved under the guidance ofa molecular motor, such as a helicase, translocase, or polymeraseagainst an electric potential difference. Molecular motors can move thepolynucleotide in a step-wise manner, normally with one or morenucleotides per step. This controlled ratcheting slows thepolynucleotide translocation through the nanopore from a native rate ofusec/nucleotide to msec/nucleotide.

A method of detection can utilize a potential difference across abarrier (e.g., a membrane). The potential difference can be an electricpotential difference, chemical potential difference, or anelectrochemical potential difference. An electric potential differencecan be imposed across the barrier (e.g., membrane) via a voltage sourcethat injects or administers current to at last one of the liquid pools.A chemical potential can be imposed across the barrier via a differencein ionic composition of the two pools. An electrochemical potentialdifference can be established by a difference in ionic composition ofthe two pools in combination with an electrical potential. The differentionic composition can be, for example, different ions in each pool ordifferent concentrations of the same ions in each pool.

The application of an electrical potential across a pore to force thetranslocation of a nucleic acid through the pore is well known in theart and can be used in accordance with the present apparatus and methods(Deamer et al., Trends Biotechnol., 18:147-151 (2000); Deamer et al.,Acc Chem Res., 35:817-825 (2002); and Li et al., Nat Mater.,2(9):611-615 (2003), each of which s incorporated herein by reference).A method set forth herein can be carried out with a voltage appliedacross a pore. The range for the voltage can be selected from 40 mV toupwards of 1 V. Typically a method set forth herein will run in therange of 100 to 200 mV. In specific instances, the method is run at 140mV or 180 mV. The voltages are not required to be static during themotion of the motor. The voltage polarity is typically applied such thatthe negatively charged nucleic acid is electrophoretically driven intothe pore. In some instances, the voltage can be reduced, or the polarityreversed, to facilitate appropriate function.

In some instances, the application of pressure differentials can beutilized to force translocation of a nucleic acid through a pore.Pressure differentials can be used in place of electrical potentials orother potential differences in methods exemplified herein.Alternatively, a pressure differential can be used in combination withelectrical potentials or other potential differences in methodsexemplified herein.

The methods of the present disclosure can produce one or more signalsthat correspond to the translocation of one or more nucleotides througha pore. Accordingly, as a target polynucleotide, or as a mononucleotideor probe derived from the target polynucleotide or mononucleotide,transits through a pore the current across the barrier changes due tobase-dependent (or probe dependent) blockage of the constriction, forexample. The signal from that change in current can be measured usingany of a variety of methods as described herein or as otherwise known inthe art. Each signal is unique to the species of nucleotide(s) (orprobe) in the pore such that the resultant signal can be used todetermine a characteristic of the polynucleotide. For example, theidentity of one or more species of nucleotide(s) (or probe) thatproduces a characteristic signal can be determined. Signals useful inthe methods of the present invention include, for example, electricalsignals and optical signals. In some aspects, the electrical signal canbe a measurement of current, voltage, tunneling, resistance, voltage,conductance; or transverse electrical measurement (see WO 2013/016486,which is incorporated herein by reference). In some aspects, theelectrical signal is an electrical current passing through a pore.

Optical signals useful in the methods of the present disclosure include,for example, fluorescence and Raman signal. The optical signals can begenerated by coupling the target nucleotide with an optical signalgenerating label, for example, a fluorescent moiety or a Raman signalgenerating moiety. For example, in dela Torre et al., Nanotechnology,23(38):385308 (2012), the optical scheme of Total Internal ReflectionFluorescence (TIRF) microscopy was employed to illuminate a wide area ofthe TiO2-coated membrane. In Soni et al., Rev Sci Instrum., 81(1):014301(2010), a method was used for integrating two single-moleculemeasurement modalities, namely, total internal reflection microscopy andelectrical detection of biomolecules using nanopores. The above tworeferences are incorporated herein.

As described herein, the nanopores (whether hybrid nanopores or tetherednanopores) can be coupled with a detection circuit, including, forexample, a patch clamp circuit, a tunneling electrode circuit, or atransverse conductance measurement circuit (such as a graphenenanoribbon, or a graphene nanogap), to record the electrical signals inmethods of the present disclosure. In addition, the pore can also becoupled with an optical sensor that detects labels, for example, afluorescent moiety or a Raman signal generating moiety, on thepolynucleotides.

Molecular motors can use the energy of nucleotide hydrolysis to drivethe translocation of a target polynucleotide through a nanopore. Ahelicase is an example in which ATP hydrolysis is the energy source forpolynucleotide translocation. For example, in one model a singlestranded polynucleotide is held in a negatively charged cleft thatseparates the two RecA domains of a helicase from a third domain. In theabsence of ATP, a bookend residue (e.g., Trp501 in HCV helicase) and aclamp residue (e.g., Arg393 in HCV helicase) prevent the single strandedpolynucleotide from sliding through a cleft. Upon ATP binding, the RecAdomains rotate, moving the positively charged Arg-clamp. The Arg-clampattracts the negatively charged single stranded polynucleotide, which inturn clears the bookend. The single stranded polynucleotide is thenrepelled by the negatively charged cleft, and the single strandedpolynucleotide translocates through the helicase until ATP ishydrolyzed. Therefore, in this exemplary model, the polynucleotidetranslocation through a helicase involves at least two steps: a firststep where the helicase binds to ATP and undergoes a conformationalchange, and a second step where ATP is hydrolyzed and the polynucleotidetranslocates through the helicase.

Other detection techniques that can be applied to an apparatus set forthherein include, but are not limited to, detecting events, such as themotion of a molecule or a portion of that molecule, particularly wherethe molecule is DNA or an enzyme that binds DNA, such as a polymerase.For example, Olsen et al, JACS 135: 7855-7860 (2013), which isincorporated herein by reference, discloses bioconjugating singlemolecules of the Klenow fragment (KF) of DNA polymerase I intoelectronic nanocircuits so as to allow electrical recordings ofenzymatic function and dynamic variability with the resolution ofindividual nucleotide incorporation events. Or, for example, Hurt etal., JACS 131: 3772-3778 (2009), which is incorporated herein byreference, discloses measuring the dwell time for complexes of DNA withthe KF atop a nanopore in an applied electric field. Or, for example,Kim et al., Sens. Actuators B Chem. 177: 1075-1082 (2012), which isincorporated herein by reference, discloses using a current-measuringsensor in experiments involving DNA captured in a α-hemolysin nanopore.Or, for example, Garalde et al., J. Biol. Chem. 286: 14480-14492 (2011),which is incorporated herein by reference, discloses distinguishingKF-DNA complexes based on the basis of their properties when captured inan electric field atop an α-hemolysin pore. Other references thatdisclose measurements involving α-hemolysin include the following, allto Howorka et al., which are incorporated herein by reference: PNAS 98:12996-13301 (2001); Biophysical Journal 83: 3202-3210 (2002); and NatureBiotechnology 19: 636-639 (2001).

U.S. Pat. No. 8,652,779 to Turner et al., which is incorporated hereinby reference, discloses compositions and methods of nucleic acidsequencing using a single polymerase enzyme complex including apolymerase enzyme and a template nucleic acid attached proximal to ananopore, and nucleotide analogs in solution. The nucleotide analogsinclude charge blockade labels that are attached to the polyphosphateportion of the nucleotide analog such that the charge blockade labelsare cleaved when the nucleotide analog is incorporated into a growingnucleic acid. According to Turner, the charge blockade label is detectedby the nanopore to determine the presence and identity of theincorporated nucleotide and thereby determine the sequence of a templatenucleic acid. U.S. Patent Publication No. 2014/0051069 which isincorporated herein by reference, is directed to constructs that includea transmembrane protein pore subunit and a nucleic acid handling enzyme.

Example I

The two major thrusts of the experimental design are to: (a) engineer,synthesize and purify the protein-nanodisc complex; and (b) assemble thecomplex on the solid state nanopore platform by electrophoretic forces.The electrophoretic driving force can be enhanced by attachingpolyelectrolyte or DNA molecule on the disc or pore-forming protein.Additional surface treatment may be required to improve the insulationon the lipid disc-solid nanopore interface.

SPECIFIC AIM #1: Demonstrate Incorporation of MspA Nanopore into LipidNanodisc

Milestones: (a) Assemble and characterize blank lipid nanodisc; (b)Engineer MspA protein with His-tag for the purpose of separation andpurification; (c) Incorporate MspA protein into lipid nanodisc, purifythe product and characterize the morphology.

Synthesize Bio-Pore Incorporated Lipid Nanodisc

Blank lipid nanodiscs are synthesized by titration of lipid and MSPratio. The experimental approach was reviewed above in connection withFIG. 3 . Biopore mutants with C-terminal His-tags are prepared. TheHis-tagged biopore mutants are added into the nanodisc precursor mixturebefore the detergent is removed so that the biopore incorporation willtake place during the lipid nanodisc self-assembly process. Excessamount of nanodisc component are used to ensure high yield ofincorporation. The nanopore-nanodisc complex is separated from emptynanodiscs by a His-tag affinity column. The whole process is monitoredand analyzed by size exclusion chromatography (SEC) and sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), andfurther characterized by liquid-phase atomic force microscopy andtransmission electron microscopy.

SPECIFIC AIM #2: Achieve >100 GΩ Seal of Lipid Nanodisc Inserted inSolid State Nanopore

Milestones: (a) Establish nanopore and electrophoresis infrastructure;(b) Electrophoretically assemble blank lipid-nanodisc onto solid statenanopore; (c) If necessary, chemically functionalize solid statenanopore to ensure >100 GΩ seal; (d) Demonstrate MspA biological poreactivity in the hybrid system.

Build Solid State Nanopore System

The fabrication methods for solid state nanopore have historicallyutilized energized beam drilling, for example, with an electron beam ina Transmission Electron Microscope (TEM)(see, for example, Storm et al.,Nature Materials 2, 537-540 (2003), incorporated herein by reference), aGa ion beam in a Focused Ion Beam (FIB))(see, for example, Patterson etal., Nanotechnology 19, 235304 (2008), incorporated herein by reference)or a Helium Ion Beam (HIB)(see, for example, Yang et al., Nanotechnology22, 285310 (2011), incorporated herein by reference). A commercialsupply of 7-10 nm nanopores will be used for initial demonstration andsystem development. Initial prototyping of the required equipment iscarried out as set forth above in relation to FIG. 6A and FIG. 6B.Electrophysiology tools such as patch-clamp amplifier andelectrochemistry workstations, and a cleanroom facility are used foradditional nanofabrication work.

Achieve Lipid Nanodisc Sealed Nanopore with >100 GΩ Resistance

Electrophoretically driven translocation of biomolecules through solidstate nanopore has been extensively studied in the past decade. The samestrategy can be applied to seal the solid state nanopore with arelatively large sized lipid nanodisc, leaving the incorporated proteinnanopore as the only pathway for ionic current and the only pathway foranalytes that are to be detected by passage through the pore. Theefficiency of sealing is tested with blank lipid nanodiscs (withoutbiopores). Since the lipid nanodisc is lightly charged, a DNA moleculederivatized with cholesterol-TEG can be utilized to improve theelectrophoresis effect of capturing lipid nanodisc. Cholesterol taggedDNA tends to bind to the lipid membrane by inserting the hydrophobiccholesterol unit into the membrane. A diagrammatic representation of ablank nanodisc having a nucleic acid tether is shown in FIG. 8 .

The electrical field guides the tether-containing nanodisc by capturingthe highly charged DNA chain. The capture probability is also affectedby nanopore size, ionic strength, voltage and viscosity, which can beadjusted to achieve desired loading. Multiple insertions of DNA tethersinto a single nanodisc are possible at this intermediate step, butshould not affect the ability to assess the sealing efficiency betweenthe nanodisc and the solid state nanopore surface.

Additional modifications may be used to achieve a >100 GΩ seal, forexample, in embodiments where a seal is not created entirely viaelectrophoretic action. If necessary, the solid-state nanopore can befunctionalized to seal interface leakage pathways. Ensuringhydrophobicity of the interface can prevent the transport of solvatedions. One technique is to coat the top surface of the solid statenanopore with cholesterol derivatives which ensure that the surface ishydrophobic, while also inserting their hydrophobic terminal groups intothe membrane. Specific surface chemistry that can be used is shown inFIG. 9 .

Another approach is local tethering of the lipid nanodisc, for example,as described in Stackmann Science 271, 43-48 (1996); Steinem et al.Biochim. Biophys. Acta, 1279, 169-180 (1996); Vallejo,Bioelectrochemistry 57, 1-7 (2002); and Avanti Polar Lipids, Inc.(October 2013), “Preparation of Liposomes”, each of which isincorporated herein by reference. Alternatively or additionally, theentire surface of the Si₃N₄ chip can be coated with a membrane, forexample, as described in Yusko et al., Nature Nanotech. 6, 253 (2011),which is incorporated herein by reference. The MspA biopore can then beinserted directly after formation of this membrane coating. Sterichindrance can be exploited to ensure that only a single porin can beinserted into each aperture, and the effective conversion of diffusionfrom 3 dimensional (i.e. in solution) to 2 dimensional (i.e. in themembrane) can provide adequate insertion efficiency. Large unilamellarvesicles with diameter of ˜100 nm can be obtained (see, for example,Avanti Polar Lipids, Inc. (October 2013), “Preparation of Liposomes”,which is incorporated herein by reference) and can bridge smaller solidstate nanopores (e.g. the ˜10 nm nanopores described in this Example).

Additionally, chemical functionalization of the solid-state nanopore maybe done to prevent any residual leakage pathway along the interfacebetween the lipid nanodisc and solid state nanopore. Thus, the topsurface of the solid state nanopore, which the lipid disc will be incontact with, can be chemically functionalized with hydrophobicself-assembled molecules. Exemplary moieties that can be used forfunctionalization are cholesterol derivatives which can insert theirhydrophobic portion into the membrane, as a result, preventing hydratedion transport along the interface. Useful surface chemistry to conjugatecholesterol derivative onto solid substrate, is exemplified in FIG. 9 .

Surface functionalization may alter the electrical characteristic of thesolid state nanopore. The effect can be beneficial by reducing thesticking events by DNA interaction with the solid surface. However, theincrease in solid state nanopore hydrophobicity may impact its transportproperties (see, for example, Powell et al., Nature Nanotechnology 6,798-802 (2011), which is incorporated herein by reference. The twoeffects can be balanced to achieve optimal system performance).

Test Biopore Activity as a Part of the Hybrid Nanopore Device

Specific Aim #2b will demonstrate the assembly of a lipidnanodisc/biopore complex onto a solid state nanopore. The electricalcharacterization of the hybrid nanopore, noise level and the stabilitycan be characterized under nucleic acid sequencing conditions. Thehybrid nanopore may possess higher noise than a conventional proteinnanopore device, mainly contributed by the capacitive coupling throughthe silicon supporting substrate. See, for example, Waggoner et al.,Journal of Vacuum Science & Technology B 29, 032206 (2011), which isincorporated herein by reference. The noise level can be optimized byresin passivation of the fluidic contact area, or adding micron thickSiO2 between the Si₃N₄ film and the Si substrate, for example, usingtechniques and materials set forth in Rosenstein et al., Nature Methods9, 487-492 (2012), which is incorporated herein by reference. Currentnanopore sequencing chemistry contains polymerase, ATP, and salt inclose to neutral pH buffer, which is not expected to affect thestability of s lipid disc/biopore complex. The performance of the hybridnanopore can be benchmarked against a conventional lipid-supportedbiopore in order to characterize its performance.

Example II

This Example describes progress on specific aim #1 of Example I. Morespecifically, protocols were established to (a) assemble andcharacterize blank lipid nanodiscs with 10-13 nm diameter, and (b)incorporate MspA nanopore proteins into lipid nanodiscs and characterizethe morphology of the complex.

Fast protein liquid chromatography (FPLC) was used to optimize nanodiscformation and MspA insertion as shown in FIG. 10 . The bottom trace(black) is for nanodiscs only, showing a well-defined elution peak froman FPLC instrument. Adding MspA to the precursor mix destabilized thereaction and resulted in a second elution peak at higher molecularweight as shown by the middle trace (red). Behavior was similar to thatof pure nanodisc synthesis in the presence of excess lipid. Although notnecessarily intending to be limited by hypothesis, the surfactantrequired to stabilize the MspA protein in solution appears to beinteracting with the nanodisc self-assembly process. By reducing theamount of lipid used during synthesis by ⅓ normal behavior was recoveredas shown by the top trace (blue).

In addition, Transmission electron microscopy (TEM) characterization wasperformed on the two elution fractions (A and B) from FIG. 10 . Thesamples were stained with uranyl acetate prior to TEM imaging to enhancecontrast. The resulting TEM images are shown in FIG. 11 . Apparentinsertion of MspA (discs with dark dots) was observed in both samples,with heavy agglomeration in the high MW fraction as expected. TEM imagesof blank nanodiscs (not shown) did not exhibit such features. Furtherconfirmation of MspA insertion can be obtained through proteinelectrophoresis, and via metal column purification of the product, asdocumented in Specific Aim #1 of Example I.

The above results confirm establishment of a protocol for synthesis ofbare nanodiscs. Furthermore, the results provide proof-of-principleconfirmation of MspA insertion in self-assembled nanodiscs.

Throughout this application various publications, patents or patentapplications have been referenced. The disclosure of these publicationsin their entireties are hereby incorporated by reference in thisapplication.

The term comprising is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

1.-104. (canceled)
 105. A method of making a detection apparatus,comprising: (a) providing a solid support comprising an array of solidstate nanopores; (b) providing a plurality of lipid nanodiscs, whereineach lipid nanodisc comprises a protein nanopore inserted in a lipidbilayer; and (c) contacting the plurality of lipid nanodiscs with thearray of solid state nanopores so that each solid state nanopore becomescovered by one lipid nanodisc.
 106. The method of claim 105, wherein (b)comprises incubating the protein nanopores with phospholipids in thepresence of a detergent; and removing the detergent from thephospholipids to form the lipid nanodiscs.
 107. The method of claim 105,wherein (c) comprises contacting the solid state nanopores with anamount of the plurality of lipid nanodiscs that exceeds an amount of thesolid state nanopores.
 108. The method of claim 105, wherein each lipidnanodisc in the plurality of lipid nanodiscs is stabilized with amembrane scaffold protein (MSP).
 109. The method of claim 105, whereineach lipid nanodisc in the plurality of lipid nanodiscs comprises nomore than one protein nanopore.
 110. The method of claim 105, whereineach lipid nanodisc in the plurality of lipid nanodiscs comprisescharged tethers attached to the lipid bilayer or to the proteinnanopore.
 111. The method of claim 110, wherein the charged tetherscomprise nucleic acids.
 112. The method of claim 110, wherein (c)comprises electrically attracting the plurality of lipid nanodiscs tothe solid state nanopores.
 113. The method of claim 105, furthercomprising (d) coupling the plurality of lipid nanodiscs with the arrayof solid state nanopores.
 114. The method of claim 113, wherein thecoupling is via a silane, a cholesterol, or a cholesterol derivative.115. The method of claim 105, wherein the plurality of lipid nanodiscsare non-contiguous from each other on a surface of the solid support.116. The method of claim 105, wherein the plurality of lipid nanodiscsform seals for at least 50% of the solid state nanopores.
 117. Themethod of claim 105, wherein the apparatus comprises a cis reservoir incontact with the array of solid state nanopores and a trans reservoir incontact with the array of solid state nanopores, and wherein the cisreservoir and the trans reservoir comprise electrodes to apply a currentthrough apertures formed by the protein nanopores.
 118. The method ofclaim 105, wherein the apparatus comprises an amplifier configured toamplify electrical signals generated at the protein nanopores.
 119. Amethod of making a nanopore detection apparatus, comprising: (a)providing a plurality of lipid nanodiscs, wherein each lipid nanodisccomprises a protein nanopore inserted in a lipid bilayer; and (b)coupling each lipid nanodisc to a solid state nanopore by contacting theplurality of lipid nanodiscs to a solid support comprising an array ofsolid state nanopores.
 120. The method of claim 119, wherein (a)comprises incubating a plurality of protein nanopores with phospholipidsin the presence of a detergent; and removing the detergent from thephospholipids to form the plurality of lipid nanodiscs.
 121. The methodof claim 119, wherein (b) comprises contacting a plurality of solidstate nanopores with an amount of the plurality of lipid nanodiscs thatexceeds an amount of a plurality of solid state nanopores.
 122. Themethod of claim 119, wherein (b) comprises electrically attracting theplurality of lipid nanodiscs to a plurality of solid state nanopores.